Project Description Drug-eluting stents (DESs) have been used widely because of their ability to prevent restenosis. A complication of DESs is development of late in-stent thrombosis at the same, if not higher, rate as the bare metal stents (BMSs). The DESs in clinical applications deliver an anti-proliferative agent, such as paclitaxel, sirolimus, or zotarolimus. These drugs inhibit not only coronary smooth muscle cells (SMCs), but also prevent proliferation of endothelial cells (ECs). Controlling the late thrombosis, which occurs between one month and one year, requires solving the fundamental problem related to the lack of endothelial cell (EC) growth in the stented area. The main objective of this project is to develop drug-eluting stents that deliver two different drugs with sequential release kinetics for controlling restenosis by inhibiting growth of smooth muscle cells (SMCs) followed by promoting EC growth. The hypothesis in this proposal is that the late in-stent thrombosis is mainly due to the lack of growth of ECs over the stented area, and thus, the release of a drug promoting the proliferation and migration of ECs in the stented area during the first few months after implantation prevents the late in-stent thrombosis. The specific aims of this project are: (1) to utilize an in vitro cell culture method that can evaluate various drugs for preventing proliferation of SMCs and promoting growth of ECs; (2) to load a set of two drugs onto a stent for sequential release with predefined release kinetics;(3) to test the efficacy of two-drug-eluting stents in the in vitro organ culture model;and (4) finally to test the two-drug- eluting stents in Ossabaw pigs prone to metabolic syndrome and excess coronary in-stent neointimal hyperplasia. The innovation in this approach is utilization of in vitro cell culture of SMCs and ECs to mimic in vivo restenosis and healing process. The in vitro cell culture approach will be used to screen various drugs and drug combinations that are most effective in preventing SMC proliferation without negatively affecting EC growth. Successful completion of this project will allow investigators in the DES area to choose the right drug and drug combinations for developing future DESs that can prevent not only restenosis, but also late thrombosis.